Arsenic compounds, their preparation methods and uses thereof

ABSTRACT

Arsenic compounds of general formula (I), their preparation methods, the pharmaceutical compositions containing the compounds, and their uses in the manufacture of medicaments for treating cancer, particularly leukemia, are disclosed.

FIELD OF THE INVENTION

The present invention relates to arsenic compounds, their preparation method and uses for cancer therapy.

BACKGROUND OF THE INVENTION

Arsenic compounds are exit in many of Chinese medicine, such as arsenic, realgar, orpiment and Arsenolite. Arsenic Compounds have been used for treating skin diseases, such as syphilis in Traditional Chinese Medicine (TCM) from long time ago. Arsenic Compounds were used for cancer therapy since 19 century; e.g Lissauer et. al used 1% potassium arsenite solution (Fowler solution) for treating leukemia. In 1971, Han, Taiyun, from The First Affiliated Hospital of Haerbing Medical College, who had developed a medicine for treating leukemia by improving a kind of TCM containing arsenic. Zhang T. D., Zhang P., et. al used “Ailing yihao” to treat chronic myeloid leukemia in 1973, which has improved the blood parameters and the clinical symptoms. However, As₂O₃ injection is highly toxic, carcinogenic, teratogenic, and mutagenic. It may cause nausea, vomit and leukopenia etc side effect (Zhao N. L. et. al., Clinical Oncology, 2005, 10(1): 87-88); Myelosuppression is another side effect after a long-term administration (Li F., et. al., Foreign Medicine Traditional Chinese Medicine Volume, 2001, 23(3): 134-138). In addition, AS₂O₃ has poor water solubility and poor bioavailability. Since there is not effective drug for clinical treating leukemia, so our effort was focused on reducing the toxicity and side effect of AS₂O₃; increasing its solubility and bioavailability, and further improving the efficiency of anti-leukemia.

CONTENT OF THE INVENTION

One of the features of the present invention is providing arsenic compounds which can effectively treat cancer, their preparation method and their compositions.

The present invention has following structure formula (I) containing arsenic compounds.

Wherein R₁ is selected independently from H, C₂-C₂₀ straight-chain or branched alkylacyl groups, or C₇-C₁₂ arylacyl groups; R₂ is selected independently from H, C₂-C₈ straight-chain or branched alkyl groups, or C₇-C₁₂ aryl groups; X could be H₂AsO₄ ⁻, HLiAsO₄ ⁻, HKAsO₄ ⁻, HNaAsO₄ ⁻, Li₂AsO₄ ⁻, K₂AsO₄ ⁻, Na₂AsO₄, CaAsO₄ ⁻, MgAsO₄ ⁻, AsO₂ ⁻, (NH₄)₂AsO₄ ⁻, (NH₄)HAsO₄ ⁻.

The present invention is preferred the following structure formula (II) containing salt formation by arsenate and carnitine derivatives.

Wherein R₁ is selected independently from H, C₂-C₂₀ straight-chain or branched alkylacyl groups, or C₇-C₁₂ arylacyl groups; R₂ is delected independently from H, C₂-C₈ straight-chain or branched alkyl groups, or C₇-C₁₂ aryl groups. Preferably, R₁ is selected from acetyl, propionyl, butyryl, valeryl, isovaleryl, dodecyl, tetradecanoyl, palmityl, stearyl, benzoyl, phenylacetyl, phenyl propionyl, phenyl butyryl, phenyl valeryl or phenyl isovaleryl; R₂ is selected from ethyl, propyl, butyl, pentyl, isopentyl, benzyl, phenethyl, phenylpropyl, phenylbutyl, phenylpentyl or phenylisopentyl.

Formula (II) includes their levoisomers, dextroisomers and racemic compound, especially preferred levoisomers.

The present invention discloses a method for preparing arsenic compounds which includes follow steps:

-   a) dissolve the compounds of formula (III) in organic solvent to     form the solution

-   b) add arsenic acid or one of the compound selected from the claim 1     to the solution of step (a), by stirring titration. -   c) Separate the salts of formula (II). -   d) Crystallize the salts of step (c) in organic solvent to obtain     the crystal.

Another characteristic of the present invention is providing anti-cancer compositions. Wherein the said cancers include liver cancer, lung cancer, pancreatic cancer, breast cancer, ovaries cancer, endometrial cancer, colon cancer, stomach cancer, kidney cancer, nasopharyngeal cancer, ovarian cancer, prostate cancer, chronic or acute leukemia, brine cancer, esophagus cancer, oral cancer, cardiac carcinoma, colon cancer, gallbladder cancer, laryngocarcinoma, gingival carcinoma, urethral carcinoma, skin cancer, colon cancer, cancer of middle ear, bone cancer, testicular cancer, cancer of endocrine system, lymphoma, primary CNS malignant lymphoma, chordoma, pituitary adenoma, or their combination of above. Preferably, the said cancer is leukemia. It contains therapeutically effective amount of arsenic compounds and pharmaceutical carriers in the present invention.

It is especially preferred that the compositions containing therapeutically effective amount of compound of formula (II) in the present invention.

The arsenic compounds and their compositions in the present invention can be made into different pharmaceutical formulations, which include oral formulations, injections and topical formulations, wherein

(1) The oral formulations include tablets, sustained release tablets, granules, hard or soft capsules, syrups, solutions, emulsion. Pharmaceutical carriers of oral formulations include fillers, disintegrating agents, adhesives, lubricants, coloring agents, flavoring agents or other conventional additives, which include starch, lactose, microcrystalline cellulose, sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, polyvinylpyrrolidone, hydroxy propyl methyl cellulose, magnesium stearate, silica and polysorbate 80, sodium lauryl sulfate.

(2) The injection formulations include sterile injective solution, sterile injective microemulsion o/w, injective sterile powder. Pharmaceutical carriers of injections include injective solvent, injective additives, which specially include injective water, injective oil-based solvent, e.g. soybean oil, injective solubilizers such as ethanol, propylene glycol, polyethylene glycol, glycerin, isotonic substances such as sodium chloride, glucose.

(3) The topical formulations are patch, suppository, cream, ointment, gel, solution, targeting formulation or suspension, wherein the said targeting preparation includes liposome, microspheres, nanoparticle, einschluesse, conjugated monoclonal antibody. Conventional pharmaceutical carriers of topical formulations are included for the topical formulations.

The drug delivery (D) methodology of the present pharmaceutical formulations include intravenous, intramuscular D, intraperitoneal D, subcutaneous D, rectal (suppository) D, intravaginal D (suppository), targeted drug D, inhalation D, gavage D, nasal D, sublingual D, micro-needle D, continuous drug delivery system, and topical D.

For topical delivery, e.g. transdermal preparation, implantable continues drug release system, the carrier for transdermal preparation includes scaffold material such as hydrophobic polysiloxane, hydrophilic polyvinyl alcohol etc; controlled release material includes polysiloxane, Ethylene—vinyl acetate copolymer etc; pressure-sensitive adhesive such as Polyisobutylene, polysiloxane and polyacrylate, Active ingredient is generally dispersed in the pressure-sensitive adhesive; Polymer materials for the implantable continues drug release system are select from the following of polylactic acid glycolic acid copolymer, polyethylene glycol polylactic acid copolymers, polylactic acid/polycaprolactone, poly[carbonate(Aden ester-co-ε-caprolactone)ester], poly-butyrolactone ester valerolactone, poly DIOXANONE (PDS), poly-3-hydroxybutyrate (PHB), poly-L-lactic acid (PLLA), polyglycolic acid (PGA), poly-ε-caprolactone (PCL), polycaprolactone/poly-lactide lactide (PCL/PLGA), hydroxyethyl methacrylate (HEMA).

The daily dosage of the composition or formulations is proscribed by clinician; it may vary according to patient's age, weight, sex and individual sensitivity to the drug, and severity of the disease etc. Generally, the dosage for adult is 0.05-5 mg active ingredient/kg per day; preferably dosage is 0.1-0.5 mg active ingredient/kg per day.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The following embodiments are used for explanation for the present invention, not for limitation to the invention.

Example 1 Preparation of Acetyl L-Carnitine Arsenate

L-acetylcarnitine (35.3 g) was weighted and put in an flask (250 ml), and 80 ml ethanol was added; they were heated and dissolved; the ethanol solution containing 31.0 g arsenate was slowly infused in 1-acetylcarnitine solution, stirred for 1 h. When it was cooled to room temperature, white crystals was precipitated, 64.4 grams of powder was obtained by grinding. Yield is 97.1%. Melting temperature is 150.6□-152.1□. ¹H-NMR (DMSO, 500 MHZ) δ: 2.28 (m, 2H), 3.16 (m, 9H), 3.35 (m, 2H), 4.36 (m, 1H), 9.51 (s, 3H); IR: 3423 cm⁻¹, 2975 cm⁻¹, 2399 cm⁻¹, 1723 cm⁻¹, 1477 cm⁻¹, 1302 cm⁻¹, 1191 cm⁻¹, 1106 cm⁻¹, 879 cm⁻¹, 769 cm⁻¹.

Example 2 Preparation of DL-Carnitine Arsenate

DL-carnitine was used instead of L-acetylcarnitine in example 1 according to the method of example 1 to get white powder, yield is 89.7%. Melting temperature is 136.1□-138.0□.

¹H-NMR (DMSO, 400 MHZ) δ: 2.67 (m, 2H), 3.27 (s, 9H), 3.49 (m, 2H), 4.67 (m, 1H); IR: 3490.3 m⁻¹, 3404.0 cm⁻¹, 3050 cm⁻¹, 2900 cm⁻¹, 1724 cm⁻¹, 1401 cm⁻¹, 1189 cm⁻¹, 1088 cm⁻¹, 975 cm⁻¹, 931 cm⁻¹.

Example 3 Preparation of L-Carnitine Arsenate

L-carnitine was used instead of L-acetylcarnitine in example 1 according to the method of example 1 to get white crystal, yield is 97.0%. Melting temperature is 152.2□-153.1□. [α]D=−14˜−16(10 g/100 ml, H2O); ¹H-NMR (CD30D, 400 MHZ) δ: 2.67 (m, 2H), 3.27 (s, 9H), 3.49 (m, 2H), 4.67 (m, 1H); IR: 3490.3 m-1, 3404.0 cm-1, 3050 cm-1, 2900 cm-1, 1724 cm-1, 1401 cm-1, 1189 cm-1, 1088 cm-1, 975 cm-1, 931 cm-1.

When it is in 20 in the X-ray diffraction patterns, the parameters are as following

10.238, d=8.6331, I/I0=36.9; 12.039, d=7.3451, I/I0=22.4; 12.500, d=7.0756, I/I0=53.7; 18.601, d=4.7662, I/I0=100.0; 19.519, d=4.5440, I/I0=35.1; 21.600, d=4.1108, I/I0=83.7; 22.021, d=4.0331, I/I0=23.6; 24.156, d=3.6806, I/I0=67.9; 25.220, d=3.5283, I/I0=27.3.

Solubility: 1 g L-carnitine arsenate was completely dissolved in 5 ml of water and 65 ml of ethanol.

Example 4 Preparation of L-Benzylcarnitine Arsenate

L-benzylcarnitine was used instead of L-acetylcarnitine in example 1 according to the method of example 1 to get white crystal, the yield is 90.1%

Example 5 Injection Preparation Methodology was Used as Following (All Contents were Measured by Weight Percentage)

active ingredient 0.3% NaCl 0.88% Add water to 100% The active ingredient and NaCl was added in water, mixed, decolored with 0.2% active carbon to get the injection.

Example 6

Tablets or Capsules Preparation Methodology were Used as Following. All contents were measured by weight percentage

the active ingredient 1.5% lactose 48% microcrystalline cellulose 45% polyvinylpolypyrrolidone 2% hydroxypropyl methylcellulose 2% magnesium stearate 1.5% Preparation: the hydroxypropyl methylcellulose was dissolved in ethanol to get granulation solution, mixed the active ingredient and excipients except magnesium stearate, added the said granulation solution to form soft material, meshed to get wet granule, dried at 60° C., meshed, ventilated, and mixed with magnesium stearate; made tablet or filled mixture into empty capsules.

Example 7

Patches preparation methodology was used as following. All contents were measured by weight percentage

active ingredient 5% polyacrylic ester 95% The active ingredient was dispersed in polyacrylic ester glue solution, degassed, lining membrane was coated with the drug-containing glue, dried, laminated with protective film, and then cut for packaging.

Example 8

Ointments preparation methodology was used as following. All contents were measured by weight percentage

active ingredient 5% lanolin 25% Vaseline 35% glycerol 20% 0.2% lauryl sodium sulfate solution 15% The active ingredient was dissolved in lauryl sodium sulfate solution, mixed with glycerol, lanolin and Vaseline.

Example 9

Collagen implants of sustained-release of L-carnitine arsenate and polyhydroxyethyl methacrylate (P-(HEMA) preparation

arseniate of L-carnitine 1.4 g collagen protein solution (20 g/L) 15 ml hydroxyethyl methylacrylate (HEMA) 6 ml ammonium persulfate (60 g/L) 1.0 ml sodium metabisulphite (12 g/L) 1.0 ml glycol 10 ml L-carnitine arsenate was mixed with all solutions above, 1 ml mixture was put into 1.5 ml plastic centrifuge tube, incubating at 37□ for 3 h; the cream white, translucent, smooth, elastic gel was the implant containing L-carnitine arsenate and P-HEMA

Example 10

Liposome preparation of L-carnitine arsenate was as following

Reverse phase evaporation was applied for the work. Lecithin and cholesterol was dissolved in 10 ml ether; L-carnitine arsenate was dissolved in 4 ml saline, mixed the two solution in a flask, stirred ultrasonically for 2 min; removed ether with vacuum rotary evaporation to get concentrating solution, added 6 ml saline, stirred ultrasonically again for 2 min; vaporized with pressure rotary evaporation to obtain liposome suspension containing L-carnitine. arsenate

Example 11 Pharmacodynamics Test of Arsenic Compounds

(1) Cell Proliferation Test

Inoculated log phase HL-60 cells (1×10⁵/ml) in to 96 well plate medium, the volume was 100 ul per well. Added test drugs with different concentration, repeat four well for each drug dose group.

culture was carried out in CO₂ incubator; Cell proliferation was detected by MTT method 48 h after culture. Transferred the solution to original well, detected the absorptivity at 492 nm by ELISA, calculated the inhibiting The proliferation rate calculation was according to the formula CI=(1-A test group/A control group)×100%. The results were showing as below:

Tablet 1 the effect of L-carnitine arsenate on HL-60 cell proliferation Group A492 Inhibiting rate Control 0.34 ± 0.005  — 7.5 uM  0.322 ± 0.009   5.2 15 uM 0.285 ± 0.011□ 16.3 30 uM 0.239 ± 0.011□ 16.3 60 uM 0.175 ± 0.008□ 48.4 120 uM  0.128 ± 0.012□ 62.5

Tablet 2 the effect of Na3AsO4 on HL-60 cell proliferation Group A492 Inhibiting rate Control 0.34 ± 0.005   — 7.5 uM  0.331 ± 0.003     2.5 15 uM 0.313 ± 0.011□□  7.9 30 uM 0.293 ± 0.015□□ 13.9 60 uM 0.257 ± 0.011□  24.5 120 uM  0.215 ± 0.006□  36.8 Wherein □P < 0.01, □□P < 0.05 It was shown in tablet 1 and tablet 2 that both drugs inhibited HL-60 cell proliferation, however, in the same concentration, the of L-carnitine arsenate is more effective; a dose dependent pattern was shown. While Na3AsO4 had less efficiency, with only 37% inhibiting rate even in 120 uM.

(2) Cell Activity Test

Inoculated log phase HL-60 cells (1×105/ml) in to 24 well plate medium, the volume was 0.5 ml per well, add drugs with different concentration. Four days after take 0.2 ml cell suspension solution, add 0.4% Trypan blue solution and 0.3 ml of PBS. Well mixed and leave them in room temperature for 10 min, count live cell numbers and total cell numbers with blood cell counting chamber. Live cell rate=live cell number/total cell number×100%

The results were in tablet 3:

Tablet 3 live cell rate after 4 d after administration (%) Drug Control 7.5 uM 15 uM 30 uM 60 uM 120 uM L-carnitine 96.08 95.45 90.32 80.42 69.97 50.29 arsenate Na3AsO4 96.08 93.11 89.06 75.36 60.06 43.11 It was shown in tablet 3, in the same concentration, the L-carnitine arseniate has less toxicity than the Na3AsO4 group, especially in higher concentration. Combined with the proliferation test, we found that the L-canitine arsenate group has obvious proliferation inhibiting effect and less cell toxicity. 

1. Arsenic compounds of formula (I) or their levoisomers, dextroisomers or racemates:

Wherein R₁ is selected independently from H, C₂-C₂₀ which is straight-chain or branched alkylacyl groups, or C₇-C₁₂ arylacyl groups; R₂ is selected independently from H, C₂-C₈ which is straight-chain or branched alkyl groups, or C₇-C₁₂ aryl groups; X is select from H₂AsO₄ ⁻, HLiAsO₄ ⁻, HKAsO₄ ⁻, HNaAsO₄ ⁻, Li₂AsO₄ ⁻, K₂AsO₄ ⁻, Na₂AsO₄ ⁻, CaAsO₄ ⁻, MgAsO₄ ⁻, AsO₂ ⁻, (NH₄)₂AsO₄ ⁻, or (NH₄)HAsO⁴⁻.
 2. Arsenic compounds of claim 1, wherein the arsenic compounds are of formula (II) or their laevoisomers, dextroisomers and racemates:

Wherein R₁ is selected independently from H, C₂-C₂₀ straight-chain or branched alkylacyl groups, or C₇-C₁₂ arylacyl groups; R₂ is selected independently from H, C₂-C₈ straight-chain or branched alkyl groups, or C₇-C₁₂ aryl groups.
 3. Arsenic compounds of claim 2, wherein the R₁ is selected from acetyl, propionyl, butyryl, valeryl, isovaleryl, dodecyl, tetradecanoyl, palmityl, stearyl, benzoyl, phenylacetyl, phenyl propionyl, phenyl butyryl, phenyl valeryl or phenyl isovaleryl; R₂ is selected from ethyl, propyl, butyl, pentyl, isopentyl, benzyl, phenethyl, phenylpropyl, phenylbutyl, phenylpentyl or phenylisopentyl; the compounds are laevoisomers, dextroisomers and racemates.
 4. Arsenic compounds of claim 1, wherein the arsenic compounds are white crystal or amorphous powder.
 5. Arsenic compounds of claim 4, wherein the arsenic crystal is L-acetylcarnitine arsenate, DL-carnitine arsenate, or L-carnitine arsenate.
 6. Arsenic compounds of claim 5, wherein the features of x-ray diffraction pattern of arsenic crystal are as following: 10.238, d=8.6331, I/I0=36.9; 12.039, d=7.3451, I/I0=22.4; 12.500, d=7.0756, I/I0=53.7; 18.601, d=4.7662, I/I0=100.0; 19.519, d=4.5440, I/I0=35.1; 21.600, d=4.1108, I/I0=83.7; 22.021, d=4.0331, I/I0=23.6; 24.156, d=3.6808, I/I0=67.9; 25.220, d=3.5283, I/I0=27.3.
 7. A pharmaceutical composition contains any compound and pharmaceutical carriers as selected from claim
 1. 8. A pharmaceutical formulation of the composition from claim 7, including oral formulations, injections and topical formulations, wherein (1) The oral formulations include tablets, sustained release tablets, granules, hard or soft capsules, syrups, solutions, and emulsion wherein pharmaceutical carriers of oral formulations include fillers, disintegrating agents, adhesives, lubricants, coloring agents, flavoring agents or other conventional additives, which include starch, lactose, microcrystalline cellulose, sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, polyvinylpyrrolidone, hydroxy propyl methyl cellulose, magnesium stearate, silica and polysorbate 80, and sodium lauryl sulfate; (2) The injection formulations include sterile injective solution, sterile injective microemulsion o/w, injective sterile powder wherein pharmaceutical carriers of injections include injective solvent, injective additives, which specially include injective water, injective oil-based solvent, e.g. soybean oil, injective solubilizers such as ethanol, propylene glycol, polyethylene glycol, glycerin, isotonic substances such as sodium chloride, and glucose; and (3) The topical formulations are patch, suppository, cream, ointment, gel, solution, targeting formulation or suspension, wherein the said targeting preparation includes liposome, microspheres, nanoparticle, einschluesse, conjugated monoclonal antibody wherein conventional pharmaceutical carriers of topical formulations are included for the topical formulations.
 9. The drug delivery (D) methodology of the present pharmaceutical formulations of claim 8 include intravenous, intramuscular D, intraperitoneal D, subcutaneous D, rectal (suppository) D, intravaginal D (suppository), targeted drug D, inhalation D, gavage D, nasal D, sublingual D, micro-needle D, continuous drug delivery system, and topical D, for topical delivery, e.g. transdermal preparation, implantable continues drug release system, the carrier for transdermal preparation includes scaffold material such as hydrophobic polysiloxane, hydrophilic polyvinyl alcohol etc; controlled release material includes polysiloxane, ethylene—vinyl acetate copolymer etc; pressure-sensitive adhesive such as polyisobutylene, polysiloxane and polyacrylate, active ingredient is generally dispersed in the pressure-sensitive adhesive; polymer materials for the implantable continues drug release system are select from the following of polylactic acid glycolic acid copolymer, polyethylene glycol polylactic acid copolymers, polylactic acid/polycaprolactone, poly[carbonate(Aden ester-co-ε-caprolactone)ester], poly-butyrolactone ester valerolactone, poly DIOXANONE (PDS), poly-3-hydroxybutyrate (PHB), poly-L-lactic acid (PLLA), polyglycolic acid (PGA), poly-ε-caprolactone (PCL), polycaprolactone/poly-lactide lactide (PCL/PLGA), and hydroxyethyl methacrylate (HEMA).
 10. Application for cancer therapy using compound selected from claim
 1. 11. The application of claim 10, wherein the cancer include liver cancer, lung cancer, pancreatic cancer, breast cancer, ovaries cancer, endometrial cancer, colon cancer, stomach cancer, kidney cancer, nasopharyngeal cancer, ovarian cancer, prostate cancer, chronic or acute leukemia, brine cancer, esophagus cancer, oral cancer, cardiac carcinoma, colon cancer, gallbladder cancer, laryngocarcinoma, gingival carcinoma, urethral carcinoma, skin cancer, colon cancer, cancer of middle ear, bone cancer, testicular cancer, cancer of endocrine system, lymphoma, primary CNS malignant lymphoma, chordoma, pituitary adenoma, or their combination above.
 12. The application of claim 11, wherein the cancer is leukemia.
 13. According to the application of claim 9, wherein the dosage of active ingredient for adult is 0.001-50 mg/kg/d.
 14. The application of claim 13, wherein the dosage for adult is 0.05-5 mg/kg/d.
 15. The application of claim 14, wherein the dosage for adult is 0.1-0.5 mg/kg/d.
 16. A method of preparation of arsenic compounds or their crystal of claim 2, which includes the following steps: a) dissolve the compounds of formula (III) in organic solvent to form solution

b) add arsenic acid or any compound selected from formula (I), well mixing by stirring and titration to the solution of step (a) c) Separate the salts of formula (II). d) Crystallize the salts of step (c) in organic solvent to obtain crystal.
 17. The method of claim 16, wherein the organic solvent is C₁₋₅ alcohol.
 18. The method of claim 17, wherein the organic solvent is ethanol.
 19. Arsenic compounds of claim 2, wherein the arsenic compounds are white crystal or amorphous powder.
 20. A pharmaceutical composition contains any compound and pharmaceutical carriers as selected from claim
 6. 21. Application for cancer therapy using compound selected from claim
 6. 22. Application for cancer therapy using composition selected from claim
 7. 23. According to the application of claim 12, wherein the dosage of active ingredient for adult is 0.001-50 mg/kg/d. 